Plasts are intracellular organelle of chlorophyll-containing plants (algae, mosses, and higher plants). Several main types of plasts can be distinguished according to their pigment content and the nature thereof: amyloplasts, which are rich in starch, chloroplasts in which the main pigments are chlorophylls, and chromoplasts for which the main pigments are keratonoids. These three categories of plasts which derive from common precursors, proplasts, have a common basic structure consisting of a double membrane enclosing the plastid stroma. In chloroplasts, there is a third membrane system forming, within the stroma, saccules called thylakoids.
Besides their essential role in photosynthesis, chloroplasts are also involved in redox reactions, for example the reduction of nitrates to ammonium. Plasts also play an essential role in the biosynthesis and/or the storage of many molecules, among which mention will be made of starch, lipids, carotenoids, most amino acids, plant hormones (abscissic acid, precursors of gibberellins, jasmonate, etc.).
Although plasts have their own genome encoding some of their proteins, a large number of the enzymes involved in the various plastid functions are encoded by the nuclear genome and imported into the plasts.
This importation is carried out via a specific mechanism, which has more particularly been studied in the case of chloroplasts (for review cf. CHEN and SCHNELL, Trends Cell Biol. 9, 222-227, 1999; KEEGSTRA and CLINE, The Plant Cell 11, 557-570, 1999; SCHLEIFF and SOLL, Planta 211, 449-456, 2000; JACKSON-CONSTAN and KEEGSTRA, Plant Physiol. 125, 1567-1676, 2001). This mechanism involves an import system in each of the two plastid membranes: in the outer membrane, the Toc (translocon at outer membrane of chloroplast) complex which comprises at least three proteins: Toc 86, 75 and 34 (KESSLER et al., Science 266, 1035-1039, 1994; PERRY and KEEGSTRA, Plant Cell 6, 93-105, 1994); in the inner membrane, the Tic (translocon at inner membrane of chloroplast) complex which comprises at least four proteins: Tic 110, 55, 22 and 20 (KESSLER and BLOBEL, Proc. Natl. Acad. Sci. 93, 7684-7689, 1996; LÜBECK et al., EMBO J. 15, 4230-4238, 1996; CALIEBE et al., EMBO J. 16, 7342-7350, 1997; KOURANOV et al., J. Cell Biol., 143, 991-1002, 1998), and also a chaperone protein in the stroma: ClpC (AKITA et al., J. Cell Biol. 136, 983-994, 1997; NIELSEN et al., EMBO J. 16, 935-946, 1997).
A major element of this mechanism is Toc75, which is the most abundant protein in the outer membrane, and forms the central pore of the translocation channel located in this membrane (SCHNELL et al., Science 266, 1007-1012, 1994; TRANEL et al., EMBO J. 14, 2436-2446, 1995). Toc75 interacts specifically with a particular sequence, called “targeting peptide” or “transit peptide”, located at the N-terminal end of the proteins imported into the plasts (MA et al., J. Cell Biol. 134, 315-327, 1996).
Many targeting peptides have been identified in the precursors of proteins targeted to the intermembrane space, the inner membrane, the stroma and, in the case of chloroplasts, to the thylakoid membrane.
Among the proteins known to have a cleavable intraplastid-targeting peptide, mention will in particular be made of proteins targeted to the intermembrane space (Tic22: KOURANOV et al., 1998, mentioned above; KOURANOV et al., J. Biol. Chem. 274, 25181-25194, 1999), proteins targeted to the inner membrane (TPT(Triose-Pi/Pi translocator): BRINK et al., J. Biol. Chem. 270, 20808-20815, 1995), proteins targeted to the stroma (ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit): DE CASTRO SILVA FILHO et al., Plant Mol. Biol. 30, 769-780, 1996; carbonic anhydrase), proteins targeted to the thylakoid membrane (LHCP (light harvesting complex): LAMPPA et al., J. Biol. Chem. 263, 14996-14999, 1988; Cfo-II: ATPase subunit) and to the thylakoid lumen (OEE1 (Oxygen Evolving Element 1): KO and CASHMORE, EMBO J. 8, 3187-3194, 1989).
These targeting peptides generally comprise between 40 and 100 amino acids, and most of them have common characteristics: they are virtually devoid of negatively charged amino acids, such as aspartic acid, glutamic acid, asparagine or glutamine; their N-terminal region is devoid of charged amino acids, and of amino acids such as glycine or proline; their central region contains a very high proportion of basic or hydroxylated amino acids, such as serine or threonine; their C-terminal region is rich in arginine and has the ability to form an amphipathic, beta-sheet secondary structure.
In the case of proteins targeted to the thylakoid lumen, the targeting peptide is bipartite and comprises additional information for crossing the thylakoid membrane (DE BOER and WEISBEEK, Biochim. Biophys. Acta. 1071, 221-253, 1991). In certain cases, this bipartite targeting peptide can also be found in proteins targeted to the thylakoid membrane (KARNAUCHOV et al., J. Biol. Chem. 269, 32871-32878, 1994).
In all cases, the targeting peptide is cleaved after importation. This cleavage is carried out by specific proteases; a protease located in the stroma (VANDERVERE et al., Proc. Natl. Acad. Sci. 92, 7177-7181, 1995), and a protease located in the lumen of the thylakoid (CHAAL et al., J. Biol. Chem. 273, 689-692, 1998) have been described.
Proteins targeted to the outer membrane do not generally comprise a cleavable signal peptide; the targeting information is contained in the mature protein (CLINE and HENRY, Annu. Rev. Cell Dev. Biol., 12, 1-26, 1996); after they have been synthesized in the cytosol, these proteins are directly incorporated into the membrane (VAN'T HOF et al, FEBS lett. 291, 350-354, 1991 and J. Biol. Chem. 268, 4037-4042, 1993; PINADUWAGE and BRUCE, J. Biol. Chem. 271, 32907-32915, 1996) by means of interactions, the nature of which remains poorly understood, with the lipid bilayer. The only known exception to date concerns the Toc75 protein or (OEP75), the targeting of which to the outer membrane requires the presence of a cleavable, bipartite N-terminal targeting peptide (TRANEL et al., 1995, mentioned above; TRANEL and KEEGSTRA, Plant Cell 8, 2093-2104, 1996).
It is known that the use of plast-targeting peptides is necessary for introducing into these plasts proteins of interest for acting on various plastid functions, in particular with the aim of improving the characteristics of plants of agronomic interest, for example the biosynthesis of lipids, of starch, of vitamins, of hormones or of proteins by said plants, or their resistance to diseases, to insects or to herbicides. For example, application EP 189707 proposes the use of cleavable targeting peptides derived from chloroplast protein precursors, and in particular of the ribulose-1, 5-bisphosphate carboxylase small subunit targeting peptide, for importing a protein of interest into chloroplasts; PCT application WO 00/12732 proposes the use of targeting peptides from various plastid proteins, for importing proteins of interest into plasts.
The plastid functions can be modified in this way, and the characteristics conferred by these modifications are very diverse.
For the purposes of nonlimiting illustration, mention may be made of:                an increase in herbicide resistance, by expression of the precursor of acetolactate synthetase (ALS), (LEE et al. EMBO J., 7, 1241-1248, 1988), of mutated acetolactate synthetase (PRESTON and POWLES, Heredity 88, 8-13, 2002); CHONG and CHOI, Biochem. Biophys. Res. Commun. 279, 462-467, 2000), or of 3-enolpyruvylshikimate-5-phosphate synthetase (EPSP synthetase) (KLEE et al., Mol. Gen. Genet., 210, 437-442, 1987);        an increase in resistance to various stresses, by expression of zeaxanthin epoxidase, (SEO et al., Trends Plant Sci., 7, 41-48, 2002), of choline monooxygenase (SHEN et al., Sheng Wu Gong Cheng Xue Bao, 17, 1-6, 2001), of the product of the ERD1_ARATH gene (KIYOSUE et al., Biochem. Biophys. Res. Commun., 15, 196, 1214-1220, 1993), of ferrochelatase (CHOW et al., J. Biol. Chem., 31, 272, 27565-27571, 1997), of omega −3 fatty acid desaturase (IBA et al., Tanpakushitsu Kakusan Koso, 39, 2803-2813, 1994; MURAKAMI et al., Science 21, 287(5452), 476-479, 2000), or glutamine synthetase (FUENTES et al., J. Exp. Bot., 52, 1071-1081, 2001);        modification of plast metabolism, so as to increase the capture of light energy (GAUBIER et al., Mol. Gen. Genet., 1, 249, 58-64, 1995), the photosynthetic and growth capacities (MIYAGAWA et al., Nature Biotech., 19, 965-969, 2001), the carotenoid content (HUGUENEY et al., Eur. J. Biochem., 1, 209, 399-407, 1992; MANN et al., Nature Biotech., 18, 888-892, 2000), or the content of various substances of interest, such as starch (PCT application WO 00/11144), essential amino acids (MUEHLBAUER et al., Plant Physiol., 106, 1303-1312, 1994), provitamin A (RÖMER et al., Nature Biotech., 18, 666-669, 2000), hormones (JOYARD et al., Plant Physiol. 118, 715-723, 1998), etc.;        overexpression and chloroplast-targeting of proteins that can be used for the purposes of bioremediation (detoxification or depollution of contaminated soils), such as ferritin, (LOBREAUX et al., Biochem. J., 15, 288(Pt 3), 931-939, 1992), proteins of the phytochelatin family (CAZALE and CLEMENS, FEBS 507, 215-219, 2001 ; TSUJI et al., BBRC 293, 653-659, 2002), etc.        
All the intraplastid-targeting peptides known in the prior art make it possible to import a protein into plasts by means of the TOC and TIC membrane import systems, as indicated above. It has been noted that the use of these peptides for targeting proteins of interest into chloroplasts may, in particular when the targeting peptide/protein of interest construct is placed under control of a strong promoter such as the 35S promoter, has the drawback of saturating these import systems, by competing with the proteins naturally targeted to the chloroplast. As a result of this, “leakages” occur, resulting, after a few days, in the presence of the protein of interest in other subcellular compartments such as the cytoplasm.
It would be desirable to have intraplastid-targeting peptides which would not depend on the TOC/TIC import system and would therefore make it possible to avoid the abovementioned drawbacks.
In previous studies aimed at identifying, by means of a proteomic approach, proteins from spinach chloroplast membrane preparations, the inventors identified, inter alia, peptides having considerable sequence similarity with a putative 41 kDa protein from Arabidopsis (TrEMBL accession number Q9SV68) (SEIGNEURIN-BERNY et al., Plant. J. 19, 217-228, 1999; FERRO et al., Electrophoresis 21, 3517-3526, 2000).